The process of facial aging occurs in a series of predictable events manifesting in the loss of both skin volume and elastic tone. Volume loss in skin, which results from many factors, including collagen breakdown, leads to the atrophy of subcutaneous fat, underlying muscle and fasciae layers of the skin, contributes to nasolabial folds, the loss of definition of the jaw line and the coarsening of skin. Loss of elastic tone in the skin results in flaccid, sagging facial tissue. To counter these effects, rejuvenation procedures attempt to correct both volume loss and tissue tone to result in the natural appearance of treated skin. These procedures may involve the use of tissue fillers, e.g., collagen injections, or the tightening provided by “lift” surgery, among other options, used individually or in combination.
A number of temporary tissue fillers, such as collagen, hyaluronan and hydroxyapatite are currently available, however, these fillers are administered through a series of injections, and provide only a temporary effect, often failing to extend beyond 12 months in the case of collagen and hyaluronan. Longer term solutions, such as hydroxyapatites (which can last 2-5 years), also include fat autografting (lasting 1 to 3 years), which uses subcutaneous adipocytes, to correct both facial volume loss and sagging to restore a more youthful appearance to facial skin.1,2,3 The after effects of injection treatments, which can last up to one week, can include swelling, redness, pain, bruising, and tenderness. Additionally, the treatments require skilled application through multiple injections, and carry a risk of infection at injection sites.
Keratinocytes are perhaps the most important cell type in providing a youthful appearance for skin. Thus, they are necessary for maintaining skin hydration and are particularly susceptible to the aging effects of environmental factors such as UV radiation since they are more constantly exposed to these factors than other skin cell types. Furthermore, recent evidence suggests that genetically determined keratinocyte factors may also contribute to the intrinsic aging process. Since keratinocytes produce paracrine factors that affect the health/functioning of fibroblasts and other dermal cells, factors that are detrimental to keratinocyte functions are therefore also detrimental to dermal cell functions.6 
Hyaluronan (also known as hyaluronate and hyaluronic acid) is a large, negatively charged glycosaminoglycan polysaccharide that is ubiquitous in the body and is present in particularly large amounts in the skin.4 Both the dermal and epidermal skin layers are rich in hyaluronan, which is present as part of the encased extracellular matrix (which also contains collagen and other proteins), where it surrounds the cells (e.g., epidermal keratinocytes and dermal fibroblasts).5 Although young skin is rich in pericellular hyaluronan in both keratinocyte and dermal layers, the amounts are reduced in both layers of aging skin, with the loss of hyaluronan from the keratinocyte layer being much more marked than in the dermal layer.6 
In youthful skin, hyaluronan efficiently encases keratinocytes in the epidermis and fibroblasts in the dermis in a jelly like capsule (the cell coat), which provides cells with adequate growth and nutrient factors that promote the collagen and elastin production typical of youthful appearing skin. In addition to hyaluronan, the primary component of the cell capsules or coats, are also found extracellular proteins and other matter, such as collagens, proteoglycans (PG) such as TSG-6, and other glycosaminoglycans. The 3-dimensional structure of hyaluronan is of a shallow helix (with high molecular weight forms being detectable as long linear chains) that can tangle on itself, thereby providing an ideal template for assembling matrices around cells. These capsules or encasements are generally retained around cells through the binding of hyaluronan to cellular receptors, e.g., CD44, RHAMM, LYVE 1, and others. As skin ages, the ability of dermal fibroblasts and keratinocytes to maintain their hyaluronan capsules diminishes, resulting in the dehydrated and sagging appearance of aging skin.5,7 Additionally, when skin is traumatized, e.g., through exposure to excessive UV radiation (sunburned), hyaluronan production by cells in the dermis is decreased, leading to an increase in hyaluronan degradation and the increased presence of hyaluronan degradation products in the skin.
Capsules of hyaluronan contain both structural matrix proteins that hydrate and protect cells, as well as nutrients, cytokines, hormones, and growth factors that are necessary for sustaining the optimal metabolic and differentiation status of cells. The ability to provide building matrices is a major factor underlying the use of hyaluronan to promote youthful skin. A second factor is the visco-elastic properties of hyaluronan, which affects the diffusion of nutrients from the vascular supply and the elasticity of skin; collectively these effects provide the texture and smoothness typical of youthful skin. A third factor is the ability of pericellular hyaluronan to provide a target for reactive oxygen species (ROS), which may be produced after exposure to UVA/B radiation, that can attack and fragment hyaluronan, in turn protecting other cellular factors from ROS-induced damage. A final factor is related to the direct biological effects of hyaluronan on keratinocyte and fibroblasts. Hyaluronan promotes both the proliferation and differentiation of keratinocytes. For example, factors such as retinoic acid, which enhance keratinocyte differentiation, also increases the pericellular hyaluronan coat. Furthermore, hyaluronan added to keratinocytes in vitro or in vivo promotes the thickness of the keratinocyte layer and enhances keratinocyte differentiation as detected by CD44 and keratin expression.5 Hyaluronan also affects fibroblast differentiation by blocking trans-differentiation into myofibroblasts, which are dermal cells that produce high levels of collagen I and have intrinsic contractile properties, both of which promote wrinkle formation.
Over time, cellular hyaluronan coats are degraded (fragmented) and are increasingly taken up by cells as part of the aging process. This degradation is due in part to the build up of oxygen free radicals that occurs over time, and to changing genetically regulated developmental program (e.g., aging) that promote the release of hyaluronidases, which break down or fragment the hyaluronan coat. The resulting fragments stimulate the uptake machinery of the cell leading to disassembly and destruction of the hyaluronan coat by cellular lysozymes. In addition to being increasingly depleted from aging skin, particularly from around keratinocytes, it has also been found that hyaluronan is depleted from areas of wrinkled skin in youth resulting from exposure to UVA/UVB radiation, steroid use or inflammation.5,6,8,9 
Hyaluronan receptor CD44 is constitutively expressed on keratinocytes and other cells in the skin, and is believed to be essential for the retention of hyaluronan around cells in layers known as “cell coats”, and for appropriate hyaluronan metabolism in the skin.5,10,11,12 The CD44 receptor is lost from skin during the aging process and following exposure to aging factors such as UV radiation or diseases/factors that cause skin atrophy.5,9 In contrast, RHAMM, a further hyaluronan receptor, that is normally not highly expressed in normal skin, has its expression is increased with exposure to UVA/B and other injuring factors. RHAMM is thought to promote the ability of CD44 to internalize/metabolize hyaluronan. Non-integral, extracellular hyaluronan binding proteins such as TSG-6, are also important in the production and retention of hyaluronan cell coats surrounding dermal cells.13 
Although hyaluronan is known to have ideal properties for use as a tissue filler in re-capturing the properties of youthful skin, the only currently available products producing their effect beneath the skin barrier rather than upon the surface of the skin, are cross-linked forms of hyaluronan that are injected to smooth facial wrinkles and to increase the volume of facial areas, such as the lips. While cross-linking of hyaluronan does enhance its retention at the injection site, these injections are not permanent and must be repeated on a regular (6-12 months) basis if the rejuvenating effect is to be preserved. However, the cross-linking of hyaluronan with itself is believed to reduce its ability to bind to cell-surface receptor proteins, a key property necessary for the encasement of cells by coats of hyaluronan. A further difficulty encountered include the difficulty to localize or “smooth” injected hyaluronan evenly under the skin. Thus, while the use of injectable hyaluronan may act as an effective temporary filler, it is not able to act in the same manner as natural hyaluronan to provide a cell-coating effect. While degradation of cross-linked injectable hyaluronan fillers could conceivably serve as a source of hyaluronan, retention of the hyaluronan is not expected owing to the known depletion of hyaluronan receptors on dermal cell surfaces and the high rate of hyaluronan degradation within the skin.
Since injectable fillers containing cross-linked hyaluronan are only administered at the site of the wrinkle or nasolabial fold, these treatments do not serve to “rejuvenate” the skin by replenishing the depleted hyaluronan levels in adjacent areas; rather, injectable treatments provide an appearance of rejuvenation by filling the depressed area. As a result, treatment with injectable dermal fillers do not aid in preventing or delaying the appearance of new wrinkles in adjacent, untreated areas, nor do they address the underlying issues of hyaluronan deficiency, and the consequent decreases in skin hydration.
Owing to its natural presence in skin, and its depletion during aging, exposure to UV radiation (sunburns and photoaging), and other skin trauma, hyaluronan is also included in many skin products in addition to its use as an injectable filler. Topically applied hyaluronan must gain entry through the hydrophobic layer of ceramide/keratin covering the outer layers of keratinocytes. However, since hyaluronan is a polyanion, it is not expected efficiently to cross the skin's keratinocyte layer. Therefore, topical hyaluronan either remains a surface treatment (e.g., traditional hyaluronan-containing skin creams) or must be injected if significant penetration into the skin is desired (e.g., in the treatment of wrinkles where cross-linked hyaluronan is injected).
It has been reported in the art that certain molecular weights of hyaluronan are able to pass through the skin barrier to some degree. Brown et al.14 indicate that hyaluronan with molecular weights of 250 and 400 kDa, formulated with the known penetration enhancers polyethylene glycol and benzyl alcohol, passes through the skin barrier. While some other reports have indicated that undefined fractions reported to contain 40-400 kDa hyaluronan can pass through mouse and human skin,5 it has also been indicated that >400 kDa native hyaluronan does not cross the skin when applied topically.5,15 Furthermore, while Brown et al. were able to demonstrate that hyaluronan was able to pass through the skin barrier, it was also clear that the proportion of hyaluronan penetrating the skin was low and that the hyaluronan rapidly passed into the bloodstream and also exhibited rapid degradation. Kaya et al.,5 have also demonstrated that topically applied hyaluronan was poorly retained in the epidermal layer, retention was transient in the dermal layer and applied hyaluronan was taken up by dermal cells and keratinocytes. Therefore, merely enabling the passage of hyaluronan through the skin barrier will not necessarily provide a useful effect; for many uses it may also be necessary for the hyaluronan to have a prolonged residence time in order to observe an effect. This is highlighted by the use of transdermal carriers to deliver hyaluronan through topical administration by Schultz et al. (U.S. Pat. No. 4,808,576). Although such applications are successful in facilitating the passage of hyaluronan through the skin barrier, the hyaluronan is not retained within the skin but instead continues to pass to the underlying joints and tendons. In addition, the requirement for a transdermal carrier, the most effective of which is DMSO, is generally not compatible with prolonged use.
Schwach-Abdellaoui and Malle (WO 2008/000260) describe compositions possessing moisturizing and anti-wrinkle properties comprising hyaluronan of two molecular weight fractions. A first, low molecular weight fraction (50 kDa), is stated to be able to pass through the skin barrier, whereas the second, higher molecular weight fraction (300 kDa) is stated to provide its more pronounced effect in diminishing skin roughness by accumulating preferentially at the surface of the skin. This use of hyaluronan, which is typical in cosmetic preparations, relies upon the use of hyaluronan as a short-lived external filler that, owing to the water soluble nature of hyaluronan is removed when the face is washed. As noted with regard to Brown et al., the proportion of hyaluronan able to pass through the skin barrier is low, and that which is able to pass through the skin barrier has a low retention rate within the skin itself. As a result, rather than relying upon topically applied hyaluronan, current biorejuvenation procedures, such as mesotherapy,16 utilize injected, non-crosslinked, hyaluronan, either alone or with other active ingredients.
Thus, there remains a need in the art to develop methods of delivering higher molecular weight fractions of hyaluronan through the skin barrier without requiring injections. Higher molecular weight hyaluronan fractions (e.g., >100 kDa) are expected to be more bioresilient and to be better able to mimic the higher molecular weight hyaluronan naturally found in the skin.
Hyaluronan fragments have recently been shown to have therapeutic effects on wound repair and physiology of normal skin. Although these fragments penetrate skin better than higher molecular weight hyaluronan, they are not retained in the extracellular compartments of skin.5 
To date, there have been a number of examples of hyaluronan, and other glycosaminoglycans, being modified through the linking of lipids for a variety of purposes. Sakurai et al. (U.S. Pat. No. 5,464,942) describes the preparation of lipidated glycosaminoglycans (including hyaluronan) where a single lipid side chain is added to either a terminal position or a single random internal position of a glycosaminoglycan. These compositions are stated to be able to inhibit the adhesion of cancer cells to blood vessel endothelial cells and their extracellular matrices.
Yerushalmi et al. (WO 2006/050246) describes the preparation of particulate lipidated glycosaminoglycan (including hyaluronan) carriers for use in the targeted drug delivery of poorly water soluble drugs. Following lipidation, the modified glycosaminoglycans are stated to self-assemble, forming spheres, wherein the hydrophilic portion of the glycosaminoglycan is on the outside surface and the hydrophobic lipid portion lies within the sheltered inner surface. Similar self-assembled nanospheres and microspheres have also been described by Margarlit and Peer (WO 03/015755), where it is taught that, depending on the amount of phospholipid bound to the hyaluronic acid, nanoparticles (˜20% of linking sites occupied) or microparticles (˜33% of linking sites occupied) could be formed.
Scott (EP 0295 092 B1), who highlights the difficulties of enabling the passage of hyaluronan through the skin, describes preparations of hyaluronic acid fragments comprising 7 to 50 monosaccharide units for topical application. Penetration through the skin barrier is aided through the addition of activity enhancers to the formulation, the use of liposomes formed from phosphatidylcholine as a delivery vehicle, or the use of a battery-operated iontophoresis patch. However, as noted by the selected range of hyaluronan preferred 7 to 25 monosaccharide units (approximately 1,300-4,700 Da) owing to the difficulty in delivering hyaluronan through the skin barrier, these formulations are unsuitable to enable the passage of higher molecular weight hyaluronan through the skin barrier.
Della Valle and Romeo (U.S. Pat. No. 4,851,521) describe the preparation of esters of hyaluronic acid for use in a variety of applications, including cosmetics and as tissue fillers, as well as for the preparation of films and threads. Although indicated for a variety of applications, there is no teaching provided that the modified hyaluronan compositions are able to transport hyaluronan through the skin barrier; rather, subcutaneous, and intradermal administrations are indicated.
Generally, the diffusion of substances through an epithelial barrier decreases sharply when the molecular weight exceeds 700 Da. Although Pinsky (WO 2009/086504) describes skin care compositions utilizing liposomes to deliver low molecular weight collagen fragments (8.5 kDa), and optionally hyaluronan, into the skin, there remains a need for methods to permit the dermal deliver of larger molecular weight collagens and elastins, as well as therapeutically useful peptides and proteins, particularly if these do not require the use and preparation of liposomes. Epithelial delivery techniques, including transdermal delivery, for peptides and proteins was recently reviewed by Antosava et al.,17 who noted that although transdermal delivery is an attractive approach for development owing to its high bioavailability, long duration of action and painless application, it is hindered by the effectiveness of the skin barrier in preventing penetration and local irritation which preclude long-term application.
Although there are reports describing the use of phospholipid-based liposomes to transfer hyaluronan across the skin barrier, a number of problems are associated with their use, most notably, a lack of stability on storage. In addition, phospholipid-based liposomes are expensive to prepare and purify on the scale required for use in cosmetic preparations.
Despite the numerous reports in the patent literature of topically-applied cosmetic compositions containing hyaluronan that are stated to facilitate the passage of hyaluronan through the skin barrier into the epidermal and dermal layers, it remains that there are no commercially available cosmetic products fulfilling these promises. In particular, there are currently no viable methods with which effectively to deliver sufficient quantities of higher molecular weight hyaluronan (e.g., >250,000 Da) to the epidermal and dermal layers of the skin using topical cosmetic formulations that allow for retention of the hyaluronan within the skin. Rather, for cosmetic purposes, demand for the development of new filler products remains directed towards injectable hyaluronan fillers, such as Hyal-System™, or the use of hyaluronan as a surface filler that resides temporarily on the skin surface.
Therefore, one object of the present invention is to provide compositions that allow for the passage of a modified hyaluronan through the skin barrier to the epidermal and dermal layers of the skin, without requiring the use of injections, liposomes or other penetration enhancers.
A further object of the present invention is to provide modified hyaluronan compositions suitable for use in dermal enhancement, hyaluronan replenishment and/or protection therapy against the signs of aging of the skin and various forms of skin atrophy.
A further object of the invention is to provide modified hyaluronan compositions suitable for use in the reduction of scarring.
A further object of the invention is to provide modified hyaluronan that can increase the degree of hyaluronan retention in the extracellular coats of dermal cells despite the depletion or absence of hyaluronan receptors, such as CD44 and RHAMM, which are believed to be essential to hyaluronan retention, and are known to be depleted in aged and damaged skin.
A further object of the invention is to provide modified hyaluronan compositions that may be used as a topically administered carrier to deliver cosmetically and pharmaceutically active therapeutic substances through the skin barrier.
A further object of the invention is to provide modified hyaluronan compositions that may be used to topically deliver proteins, polypeptides and other large biomacromolecules (molecular weights of 700 Da to about 400-500 kDa) through the skin barrier.
A further objection of the invention is to provide modified glycosaminoglycan compositions that are able to penetrate the skin barrier for use in replenishing the levels of glycosaminoglycans within the skin, acting as hyaluronan mimetics, delivering cosmetically and therapeutically active substances, and delivering polypeptides, proteins and other large biomolecules.
A further object of the invention is to provide methods of manufacturing the above described modified glycosaminoglycan compositions wherein an activating agent is used as the limiting reagent to control the amount of lipid that is covalently bound to the glycosaminoglycan.
Further and other objects of the invention will be realized from the following Summary of the Invention, the Discussion of the Invention and the embodiments and Examples thereof.